A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis

Jiang, J., Wang, X. D., He, K., Li, X., Chen, C. M., Sayeski, P. P., Waters, M. J. and Frank, S. J. (2004) A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis. Molecular Endocrinology, 18 12: 2981-2996.


Author Jiang, J.
Wang, X. D.
He, K.
Li, X.
Chen, C. M.
Sayeski, P. P.
Waters, M. J.
Frank, S. J.
Title A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis
Journal name Molecular Endocrinology   Check publisher's open access policy
ISSN 0888-8809
Publication date 2004
Sub-type Article (original research)
DOI 10.1210/me.2004-0102
Volume 18
Issue 12
Start page 2981
End page 2996
Total pages 16
Place of publication Baltimore
Publisher The Endocrine Society
Collection year 2004
Language eng
Subject C1
270103 Protein Targeting and Signal Transduction
730105 Endocrine organs and diseases (incl. diabetes)
Abstract The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.
Keyword Endocrinology & Metabolism
Jak2 Tyrosine Kinase
Binding-protein
Extracellular Domain
Erythropoietin Receptor
Cytoplasmic Domain
Disulfide Linkage
Cleavage Site
Im-9 Cells
Dimerization
Antagonist
Q-Index Code C1

 
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